Funds are requested to purchase integrated multiphoton laser scanning microscope system for imaging living brain tissue. This non-linear optical (NLO) system will be based on the Zeiss LSM 510 scan system and an Axioskop 2Plus FS motorized upright microscope. The primary fluorescent excitation source will be a 5 watt Coherent titanium-sapphire femtosecond laser. This system will be optimized for simultaneous NLO imaging and patch clamp electrophysiology in living brain slices, nematodes and semi-intact spinal cord preparations. The proposed instrument will be housed and administered by the Neuroscience Imaging Center (NIC). The NIC currently maintains two other major microscope systems and has a full-time technician available for user training and minor maintenance. Projects from ten users (four major and six minor) are described. All proposed projects exploit the unique advantages of this upright NLO system including reduced phototoxicity, deep optical sectioning through thick living preparations, and simultaneous whole cell patch clamp electrophysiology. Dr. Ben Strowbridge will use NLO imaging to record unitary presynaptic calcium transients in olfactory bulb and dentate gyrus brain slices. Dr. Lynn Landmesser will use NLO imaging to study local circuits in the developing spinal cord in chick embryos and transgenetic mice. Dr. Stefan Herlitze plans to use this multiphoton microscope to study calcium channel function in living C. elegans. Dr. David Friel will use NLO imaging to study subcellular calcium handling in Purkinje neurons in cerebellar brain slices from normal and mutant mice.